Anti-phf-tau antibodies and their uses

ABSTRACT

Anti-PHF-tau antibodies and methods of making and using them are disclosed.

This application claims the benefit of U.S. Provisional Application No.61/577,817, filed 20 Dec. 2011, the entire content of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to anti-PHF-tau antibodies, and methods ofmaking and using them.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (AD) is a degenerative brain disorder characterizedclinically by progressive loss of memory, cognition, reasoning, judgmentand emotional stability that gradually leads to profound mentaldeterioration and ultimately death. AD is a very common cause ofprogressive mental failure (dementia) in aged humans and is believed torepresent the fourth most common medical cause of death in the UnitedStates. AD has been observed in ethnic groups worldwide and presents amajor present and future public health problem.

The brains of individuals with AD exhibit characteristic lesions termedsenile (or amyloid) plaques, amyloid angiopathy (amyloid deposits inblood vessels) and neurofibrillary tangles. Large numbers of theselesions, particularly amyloid plaques and neurofibrillary tangles ofpaired helical filaments, are generally found in several areas of thehuman brain important for memory and cognitive function in patients withAD.

The main protein component of the neurofibrillary degeneration in AD andseveral other neurodegenerative diseases is a hyperphosphorylated formof the microtubule associated protein tau. Developing therapeuticspreventing or clearing tau aggregation has been of interest for manyyears but candidate drugs, including anti-aggregation compounds andkinase inhibitors, have only just entered in clinical testing (Brunden,et al. Nat Rev Drug Discov 8:783-93, 2009)

Recently, preclinical evidence has been produced in transgenic tau mousemodels that active and passive immunization for tau can have therapeuticpotential (Chai, et al. J Biol Chem 286:34457-67, 2011, Boutajangout, etal. J Neurochem 118:658-67, 2011, Boutajangout, et al. J Neurosci30:16559-66, 2010, Asuni, et al. J Neurosci 27:9115-29, 2007). Atauopathy transmission and spreading hypothesis has recently beendescribed and is based on the Braak stages of tauopathy progression inhuman brain and tauopathy spreading after tau aggregate injections inpreclinical tau models (Frost, et al. J Biol Chem 284:12845-52, 2009,Clavaguera, et al. Nat Cell Biol 11:909-13, 2009). Thus, there is a needfor therapeutics to prevent tau aggregation and tauopathy progression totreat AD and other neurodegenerative diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows competition of labeled AT8 by various anti-tau antibodies.

FIG. 2 shows competition of labeled PT1 by various anti-tau antibodies.

FIG. 3 shows competition of labeled PT3 by various anti-tau antibodies.

FIG. 4 shows competition of labeled AT100 by various anti-tauantibodies.

FIG. 5 shows competition of labeled HT7 by various anti-tau antibodies.

FIG. 6 (A) Analysis of phosphorylated tau in brainstem homogenates(fraction P1) of 5 month old female P301L transgenic animals treatedwith saline, mouse IgG1, PT3 or AT8 as indicated in the figure, or fromnon-treated non-transgenic animals (B6). ELISA was done using AT8 (leftpanel) or AT100 (right panel) as capture antibodies followed bybiotinylated-HT7 and avidin-HRP. ELISA signals are plotted as a relativeamount of AD brain homogenate (ng/ml) providing the same ELISA signal asan average samples from a non-transgenic animal (B6). Data are plottedindividually together with mean+/−S.D. p values for differences betweenPT3- and IgG1-treated animals are indicated. (B) Western blot ofbrainstem homogenates fraction P1 from IgG1- or PT3-treated animalsusing AT100. Signal from homogenates of 10 animals treated with IgG1(IgG1-1 to IgG1-10) and 7 animals treated with PT3 (PT3-1, PT3-2, PT3-7to PT3-10 are shown. Actin was used as a loading control.

FIG. 7 Levels of total tau in sarcosyl soluble (pT4 soluble), total tauin insoluble (pT4 insoluble) and phosphorylated tau in insoluble (AT8insoluble) cortex homogenates derived from 5-month old female P301Ltransgenic mice treated with PT3 or isotype control (IgG) as indicatedin the figure. Levels are shown as a measure of a singal from ELISAplotted individually together with mean+/−SD. The sample 1m inj is apositive control sample derived from a P301L mouse brain injected with atau aggregate.

SUMMARY OF THE INVENTION

One aspect of the invention is an isolated antibody that binds PHF-taucomprising an antigen-binding site of a heavy chain variable region (VH)of SEQ ID NO:35 or 37, and an antigen-binding site of a light chainvariable region (VL) of SEQ ID NO:36 or 38.

Another aspect of the invention is an isolated antibody that bindsPHF-tau comprising certain heavy chain and light chain complementaritydetermining regions.

Another aspect of the invention is an isolated antibody that bindsPHF-tau comprising an antigen-binding site of a VH of SEQ ID NO:35 andan antigen-binding site of a VL of SEQ ID NO: 36.

Another aspect of the invention is an isolated antibody that bindsPHF-tau comprising an antigen-binding site of a VH of SEQ ID NO:37 andan antigen-binding site of a VL of SEQ ID NO: 38.

Another aspect of the invention is an isolated antibody or fragment thatcompetes for PHF-tau binding with a monoclonal antibody comprising anantigen-binding site of a VH of SEQ ID NO: 35 and an antigen-bindingsite of a VL of SEQ ID NO: 36, or an antigen-binding site of a VH of SEQID NO: 37 and an antigen-binding site of a VL of SEQ ID NO: 38.

Another aspect of the invention is polynucleotides encoding theantibodies of the invention or fragments thereof.

Another aspect of the invention is a vector comprising thepolynucleotides of the invention.

Another aspect of the invention is a host cell comprising the vector ofthe invention.

Another aspect of the invention is a method of making an antibody thatbinds PHF-tau comprising culturing the host cell of the invention andrecovering the antibody produced by the host cell.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibodies” as used herein is meant in a broad sense andincludes immunoglobulin or antibody molecules including polyclonalantibodies, monoclonal antibodies including murine, human,human-adapted, humanized and chimeric monoclonal antibodies and antibodyfragments.

In general, antibodies are proteins or peptide chains that exhibitbinding specificity to a specific antigen. Antibody structures are wellknown. Immunoglobulins can be assigned to five major classes, namelyIgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domainamino acid sequence. IgA and IgG are further sub-classified as theisotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Antibody light chains ofany vertebrate species can be assigned to one of two clearly distincttypes, namely kappa (κ) and lambda (λ), based on the amino acidsequences of their constant domains.

The term “antibody fragments” means a portion of an intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂ and Fvfragments, CDR, antigen-binding site, heavy or light chain variableregion, diabodies, single chain antibody molecules and multispecificantibodies formed from at least two intact antibodies or fragmentsthereof.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by “antigen-binding sites”. Theantigen-binding sites are defined using various terms as follows: (i)Complementarity Determining Regions (CDRs) are based on sequencevariability (Wu and Kabat J Exp Med 132:211-50, 1970). Generally, theantigen-binding site has three CDRs in each variable region (HCDR1,HCDR2 and HCDR3 in heavy chain variable region (VH) and LCDR1, LCDR2 andLCDR3 in light chain variable region (VL)) (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md., 1991). (ii) The term“hypervariable region”, “HVR”, or “HV” refers to the regions of anantibody variable domain which are hypervariable in structure as definedby Chothia and Lesk (Chothia and Lesk J Mol Biol 196:901-17, 1987).Generally, the antigen-binding site has three hypervariable regions ineach VH (H1, H2, H3) and VL (L1, L2, L3). Chothia and Lesk refer tostructurally conserved HVs as “canonical structures”. Numbering systemsas well as annotation of CDRs and HVs have recently been revised byAbhinandan and Martin (Abhinandan and Martin Mol Immunol 45:3832-9,2008). (iii) Another definition of the regions that form theantigen-binding site has been proposed by Lefranc (Lefranc, et al. DevComp Immunol 27:55-77, 2003) based on the comparison of V domains fromimmunoglobulins and T-cell receptors. The International ImMunoGeneTics(IMGT) database (http: //www_imgt_org) provides a standardized numberingand definition of these regions. The correspondence between CDRs, HVsand IMGT delineations is described in Lefranc et aL, supra. (iv) Theantigen-binding site can also be delineated based on SpecificityDetermining Residue Usage (SDRU) (Almagro J Mol Recognit 17:132-43,2004), where Specificity Determining Residues (SDR), refers to aminoacid residues of an immunoglobulin that are directly involved in antigencontact.

“Framework” or “framework sequence” are the remaining sequences withinthe variable region of an antibody other than those defined to beantigen-binding site sequences. Because the exact definition of anantigen-binding site can be determined by various delineations asdescribed above, the exact framework sequence depends on the definitionof the antigen-binding site.

The term “monoclonal antibody” (mAb) as used herein means an antibody(or antibody fragment) obtained from a population of substantiallyhomogeneous antibodies. Monoclonal antibodies are highly specific,typically being directed against a single antigenic determinant.

The term “epitope” as used herein means a portion of an antigen to whichan antibody specifically binds. Epitopes usually consist of chemicallyactive (such as polar, non-polar or hydrophobic) surface groupings ofmoieties such as amino acids, phosphorylated amino acids orpolysaccharide side chains and can have specific three-dimensionalstructural characteristics, as well as specific charge characteristics.An epitope can be linear in nature or can be a discontinuous epitope,e.g., a conformational epitope, which is formed by a spatialrelationship between non-contiguous amino acids of an antigen ratherthan a linear series of amino acids. A conformational epitope includesepitopes resulting from folding of an antigen, where amino acids fromdiffering portions of the linear sequence of the antigen come in closeproximity in 3-dimensional space.

Tau is an abundant central and peripheral nervous system protein havingmultiple well known isoforms. In the human CNS, six major tau isoformsranging in size from 352 to 441 exist due to alternative splicing(Hanger, et al. Trends Mol Med 15:112-9, 2009). These isoforms differfrom each other by the regulated inclusion of 0-2 N-terminal inserts,and 3 or 4 tandemly arranged microtubule-binding repeats, and arereferred to as 0N3R (SEQ ID NO:1), 1N3R (SEQ ID NO:2), 2N3R (SEQ IDNO:3), 0N4R (SEQ ID NO:4), 1N4R (SEQ ID NO:5) and 2N4R (SEQ ID NO:6).The term “control tau” as used herein refers to the tau isoform of SEQID NO:6 that is devoid of phosphorylation and other post-translationalmodifications.

Tau binds microtubules and regulates transport of cargo through cells, aprocess that can be modulated by tau phosphorylation. In AD and relateddisorders abnormal phosphorylation of tau is prevalent and thought toprecede and/or trigger aggregation of tau into fibrils, termed pairedhelical filaments (PHF). The major constituent of PHF ishyperphosphorylated tau. The term “paired helical filament-tau” or“PHF-tau” as used herein refers to well known tau aggregates in pairedhelical filaments. Two major regions in PHF structure are evident inelectron microscopy, the fuzzy coat and the core filament; the fuzzycoat being sensitive to proteolysis and located outside of thefilaments, and the protease resistant core of filaments forming thebackbone of PHFs (Wischik, et al. Proc Natl Acad Sci USA 85:4884-8,1988).

“Antibodies that bind PHF-tau” as used herein refers to antibodies thatbind PHF-tau as assessed on western blot. Typically, antibody binding toPHF-tau can be assessed after Coomassie stain of about 500 ng of PHF-tauafter 1 hour blocking in 5% (w/v) nonfat dry milk (NFDM) TBS-T, 0.05%Tween-20. Antibodies that bind PHF-tau optionally do not bind controltau (SEQ ID NO:6) as measured by western blot when tested under antigenloading condition where both control tau and PHF-tau is detected equallyby tau antibodies that have no preference for PHF-tau epitopes (e.g.antibody HT7, (ThermoScientific, Rockford, Ill.) (Mercken, et al. JNeurochem 58:548-53, 1992). Such exemplary antigen loading conditionsare 500 ng PHF-tau and 200 ng control tau.

Conventional well known one and three-letter amino acid codes are usedherein.

Compositions of Matter

The present invention relates to anti-PHF-tau antibodies and uses ofsuch antibodies. Such anti-PHF-tau antibodies may have the properties ofbinding a phosphorylated epitope on PHF-tau or binding to anon-phosphorylated epitope on PHF-tau. Anti-PHF-tau antibodies may beuseful as therapeutics, and as research or diagnostic reagents to detectPHF-tau in biological samples, for example in tissues or cells.

One embodiment of the invention is an isolated antibody that bindsPHF-tau comprising an antigen-binding site of a heavy chain variableregion (VH) of SEQ ID NO:35 or 37, or an antigen-binding site of a lightchain variable region (VL) of SEQ ID NO:36 or 38. Table 1 showsantigen-binding site residues of exemplary antibodies of the inventiondefined according to Kabat or Chothia as well as exemplary heavy andlight chain variable regions.

In another embodiment, the antigen-binding site of the VH of theantibodies of the invention comprises heavy chain complementaritydetermining regions (CDRs) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) of SEQ IDNOs:7, 8 and 9 or 13, 14 and 15, respectively, or the antigen-bindingsite of the VL of the antibodies of the invention comprises light chainCDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs:10, 11 and 12 or16, 17 and 18, respectively.

Another embodiment of the invention is an isolated antibody that bindsPHF-tau comprising an antigen-binding site of a heavy chain variableregion (VH) of SEQ ID NO:35 or 37, and an antigen-binding site of alight chain variable region (VL) of SEQ ID NO:36 or 38.

In another embodiment, the antigen-binding site of the VH of theantibodies of the invention comprises heavy chain complementaritydetermining regions (CDRs) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) of SEQ IDNOs:7, 8 and 9 or 13, 14 and 15, respectively, and the antigen-bindingsite of the VL of the antibodies of the invention comprises light chainCDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs:10, 11 and 12 or16, 17 and 18, respectively.

TABLE 1 Sequence SEQ ID  name NO: Sequence PT1 HCDR1,  7 SSWMG KabatPT1 HCDR2,  8 DILPGSGGTNYNERFKG Kabat PT1 HCDR3,  9 SYYDYDRFAN KabatPT1 LCDR1,  10 RSSESLLHSNGNTYLY Kabat PT1 LCDR2,  11 RMSNLAS KabatPT1 LCDR3,  12 MQYLEYPLT Kabat PT3 HCDR1,  13 SYAMS Kabat PT3 HCDR2,  14SISKGGNTYYPNSVKG Kabat PT3 HCDR3,  15 GWGDYGWFAY Kabat PT3 LCDR1,  16KASQDINRYLN Kabat PT3 LCDR2,  17 RANRLLD Kabat PT3 LCDR3,  18 LQYDEFPLTKabat PT1 HCDR1,  19 GYTFSSS Chothia PT1 HCDR2,  20 LPGSGG ChothiaPT1 HCDR3,  21 SYYDYDRFA Chothia PT1 LCDR1,  22 SESLLHSNGNTY ChothiaPT1 LCDR2,  23 RMS Chothia PT1 LCDR3,  24 YLEYPL Chothia PT3 HCDR1,  25GFTFSSY Chothia PT3 HCDR2, 26 SKGGN Chothia PT3 HCDR3,  27 GWGDYGWFAChothia PT3 LCDR1,  28 SQDINRY Chothia PT3 LCDR2,  29 RAN ChothiaPT3 LCDR3,  30 YDEFPL Chothia PT1 VH 35 QVQLQQSGTELMKPGASVKISCKATGYTFSSSWMGWVKQRPGHGLEWIGDILPGSGGTNYNE RFKGKASFTAETSSNTAYMQLSSLTSEDSAVYYCVRSYYDYDRFANWGQGTLVTVSA PT1 VL 36 DIVMTQAAPSVPVTPGESVSISCRSSESLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASG VPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQYLEYPLTFGAGTKLELK PT3 VH 37 EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYAMSWVRQNPEKRLEWVASISKGGNTYYPNS VKGRFTISRDNARNILYLQMSSLRSEDTALYYCARGWGDYGWFAYWGQVTLVTVSA PT3 VL 38 DIKMTQSPSSMYASLGERVTITCKASQDINRYLNWFQQKPGKSPKTLIYRANRLLDGVPSRF SGSGSGQDYSLTISSLDYEDMGIYYCLQYDEFPLTFGDGTKLELK

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, one from a heavy and one from a light chain, a skilledartisan will recognize that alternative embodiments may comprise singleheavy or light chain variable regions. The single variable region can beused to screen for variable domains capable of forming a two-domainspecific antigen-binding fragment capable of, for example, binding toPHF-tau. The screening may be accomplished by phage display screeningmethods using for example hierarchical dual combinatorial approachdisclosed in PCT Publ. No. WO92/01047. In this approach, an individualcolony containing either a H or L chain clone is used to infect acomplete library of clones encoding the other chain (L or H), and theresulting two-chain specific antigen-binding domain is selected inaccordance with phage display techniques as described.

Another embodiment of the invention is an isolated antibody that bindsPHF-tau comprising an antigen-binding site of a heavy chain variableregion (VH) of SEQ ID NO:35 and an antigen-binding site of a light chainvariable region (VL) of SEQ ID NO: 36.

Another embodiment of the invention is an isolated antibody that bindsPHF-tau comprising an antigen-binding site of a heavy chain variableregion (VH) of SEQ ID NO:37 and an antigen-binding site of a light chainvariable region (VL) of SEQ ID NO: 38.

Another embodiment of the invention is an isolated antibody that bindsPHF-tau comprising heavy chain complementarity determining regions(CDRs) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs:7, 8 and 9,respectively, and light chain CDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) ofSEQ ID NOs:10, 11 and 12, respectively.

Another embodiment of the invention is an isolated antibody that bindsPHF-tau comprising heavy chain complementarity determining regions(CDRs) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs:13, 14 and 15,respectively, and light chain CDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) ofSEQ ID NOs:16, 17 and 18, respectively.

In any of the preceding embodiments, the isolated antibody that bindsPHF-tau may be humanized.

Antibodies of the present invention can be produced by a variety oftechniques, for example by the hybridoma method (Kohler and MilsteinNature 256:495-7, 1975). Chimeric mAbs containing a light chain andheavy chain variable region derived from a donor antibody (typicallymurine) in association with light and heavy chain constant regionsderived from an acceptor antibody (typically another mammalian speciessuch as human) can be prepared by the method disclosed in U.S. Pat. No.4,816,567. CDR-grafted mAbs having CDRs derived from a non-human donorimmunoglobulin (typically murine) and the remainingimmunoglobulin-derived parts of the molecule being derived from one ormore human immunoglobulins can be prepared by techniques known to thoseskilled in the art such as that disclosed in U.S. Pat. No. 5,225,539.Fully human mAbs lacking any non-human sequences can be prepared fromhuman immunoglobulin transgenic mice by techniques referenced in(Lonberg, et al. Nature 368:856-9, 1994, Fishwild, et al. Nat Biotechnol14:845-51, 1996, Mendez, et al. Nat Genet 15:146-56, 1997). Human mAbscan also be prepared and optimized from phage display libraries(Knappik, et al. J Mol Biol 296:57-86, 2000, Krebs, et al. J ImmunolMethods 254:67-84, 2001, Shi, et al. J Mol Biol 397:385-96, 2010).

Antibody humanization can be accomplished using well known methods, suchas specificity determining residues resurfacing (SDRR) (U.S. Publ. No.2010/0261620), resurfacing (Padlan et al. Mol. Immunol. 28:489-98,1991), super humanization (Int. Pat. Publ. No. WO04/006955) and humanstring content optimization (U.S. Pat. No. 7,657,380). Human frameworksequences useful for grafting/humanization can be selected from relevantdatabases by those skilled in the art. The selected frameworks mayfurther be modified to preserve or enhance binding affinity bytechniques such as those disclosed in Queen et al. (Queen, et al. ProcNatl Acad Sci USA 86:10029-33, 1989) or in U.S. Publ. No. 2011/0092372.

Preparation of PHF-tau to be used as an antigen for immunization orisolating antibodies from phage display libraries can be done using anysuitable technique. In an exemplary method, PHF-tau is isolated frombrains of patients having AD using well know protocols, such asdescribed in Greenberg and Davies (Greenberg and Davies Proc Natl AcadSci USA 87:5827-31, 1990). PHF-tau may be isolated from the postmortemcortex of an Alzheimer patient. The isolated PHF-tau is characterizedfor its purity and hyperphosphorylation status with antibodies known toreact with PHF-tau. In a typical PHF-tau preparation, thehyperphosphorylated bands migrating at about 60, 64, 68 and 72 kDa inwestern blot (Spillantini and Goedert Trends Neurosci 21:428-33, 1998)are detected by an AT8 antibody that specifically bindshyperphosphorylated PHF-tau but not dephoshporylated PHF-tau.

Antibodies of the present invention may have the characteristics of notbinding control tau of SEQ ID NO:6. Such antibodies may be generatedusing methods described above and testing the antibodies for their lackof binding to control tau in western blots followed by Coomassie stainas described above. Control tau may be recombinantly expressed andpurified using standard methods. Exemplary antibodies binding PHF-taubut not control tau are antibodies PT1 and PT3. The antibodies of theinvention may further be evaluated for their specificity for exampleusing immunohistochemistry on control and AD brain slices.

The antibodies of the invention may have affinities towards PHF-tau witha dissociation constant (K_(D)) less than or equal to about 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹ or 10⁻¹² M. The affinity of a given molecule forPHF-tau can be determined experimentally using any suitable method. Suchmethods may utilize Biacore, ProteOn or KinExA instrumentation, ELISA orcompetitive binding assays known to those skilled in the art.

Another aspect of the invention is an isolated antibody or fragment thatcompetes for PHF-tau binding with a monoclonal antibody comprising anantigen-binding site of a heavy chain variable region (VH) of SEQ IDNO:35 and an antigen-binding site of a light chain variable region (VL)of SEQ ID NO:36, or an antigen-binding site of a heavy chain variableregion (VH) of SEQ ID NO:37 and an antigen-binding site of a light chainvariable region (VL) of SEQ ID NO:38.

Competition between binding to PHF-tau can be assayed in vitro usingwell known methods. For example, binding of MSD Sulfo-Tag™NHS-ester-labeled antibody to PHF-tau in the presence of an unlabeledantibody can be assessed using immunoassay followed byelectrochemiluminescence detection.

Several well known methodologies in addition to competitive binding canbe employed to determine the binding epitope of the antibodies of theinvention. For example, when the structures of both individualcomponents are known, in silico protein-protein docking can be carriedout to identify compatible sites of interaction. Hydrogen-deuterium(H/D) exchange can be carried out with the antigen and antibody complexto map regions on the antigen that may be bound by the antibody. Segmentand point mutagenesis of the antigen can be used to locate amino acidsimportant for antibody binding. Co-crystal structure of antibody-antigencomplex is used to identify residues contributing to the epitope andparatope.

Antibodies of the invention may be monoclonal antibodies of the IgG,IgD, IgA or IgM isotypes. Antibodies of the invention may be bispecificor multispecific. An exemplary bispecific antibody may bind two distinctepitopes on PHF-tau or may bind PHF-tau and amyloid beta (Aβ). Anotherexemplary bispecific antibody may bind PHF-tau and an endogenousblood-brain barrier transcytosis receptor such as insulin receptor,transferring receptor, insulin-like growth factor-1 receptor, andlipoprotein receptor. An exemplary antibody is of IgG1 type.

Immune effector properties of the antibodies of the invention may beenhanced or silenced through Fc modifications by techniques known tothose skilled in the art. For example, Fc effector functions such as C1qbinding, complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. can be providedand/or controlled by modifying residues in the Fc responsible for theseactivities. Pharmacokinetic properties could also be enhanced bymutating residues in the Fc domain that extend antibody half-life(Strohl Curr Opin Biotechnol 20:685-91, 2009).

Additionally, antibodies of the invention can be post-translationallymodified by processes such as glycosylation, isomerization,deglycosylation or non-naturally occurring covalent modification such asthe addition of polyethylene glycol moieties (pegylation) andlipidation. Such modifications may occur in vivo or in vitro. Forexample, the antibodies of the invention can be conjugated topolyethylene glycol (PEGylated) to improve their pharmacokineticprofiles. Conjugation can be carried out by techniques known to thoseskilled in the art. Conjugation of therapeutic antibodies with PEG hasbeen shown to enhance pharmacodynamics while not interfering withfunction (Knight, et al. Platelets 15:409-18, 2004, Leong, et al.Cytokine 16:106-19, 2001, Yang, et al. Protein Eng 16:761-70, 2003).

Another embodiment of the invention is an isolated polynucleotideencoding the antibodies of the invention or their complement, orfragments thereof. Exemplary isolated polynucleotides arepolynucleotides encoding polypeptides comprising an immunoglobulin heavychain CDRs HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs:7, 8 and 9 or 13,14 and 15, respectively, or polypeptides comprising an immunoglobulinlight chain CDRs LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs:10, 11 and12 or 16, 17 and 18, respectively, and polynucleotides having a sequenceshown in SEQ ID NOs:31-34, encoding antibody variable regions of theinvention. Other polynucleotides which, given the degeneracy of thegenetic code or codon preferences in a given expression system, encodethe antibodies of the invention are also within the scope of theinvention. The isolated nucleic acids of the present invention can bemade using well known recombinant or synthetic techniques. DNA encodingthe monoclonal antibodies is readily isolated and sequenced usingmethods known in the art. Where a hybridoma is produced, such cells canserve as a source of such DNA. Alternatively, using display techniqueswherein the coding sequence and the translation product are linked, suchas phage or ribosomal display libraries, the selection of the binder andthe nucleic acid is simplified. After phage selection, the antibodycoding regions from the phage can be isolated and used to generate wholeantibodies, including human antibodies, or any other desired antigenbinding fragment, and expressed in any desired host, including mammaliancells, insect cells, plant cells, yeast, and bacteria.

Another embodiment of the invention is a vector comprising at least onepolynucleotide of the invention. Such vectors may be plasmid vectors,viral vectors, transposon based vectors or any other vector suitable forintroduction of the polynucleotides of the invention into a givenorganism or genetic background by any means.

Another embodiment of the invention is a host cell comprising any of thepolynucleotides of the invention. Such host cells may be eukaryoticcells, bacterial cells, plant cells or archeal cells. Exemplaryeukaryotic cells may be of mammalian, insect, avian or other animalorigins. Mammalian eukaryotic cells include immortalized cell lines suchas hybridomas or myeloma cell lines such as SP2/0 (American Type CultureCollection (ATCC), Manassas, Va., CRL-1581), NS0 (European Collection ofCell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO(ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. Anexemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Otheruseful cell lines include those derived from Chinese Hamster Ovary (CHO)cells such as CHO-K1 SV (Lonza Biologics), CHO-K1 (ATCC CRL-61,Invitrogen) or DG44.

Another embodiment of the invention is a method of making an antibodythat binds PHF-tau comprising culturing a host cell of the invention andrecovering the antibody produced by the host cell. Methods of makingantibodies and purifying them are well known in the art.

Methods of Treatment

Anti-PHF-tau antibodies of the invention or fragments thereof, includingFab, (Fab′)2, scFv fragments, or antibodies comprising antigen-bindingsites of the antibodies of the invention can be used to treat, reduce orprevent symptoms in patients having a neurodegenerative disease thatinvolves pathological aggregation of tau within the brain, such aspatients suffering from AD or any other tauopathy. While not wishing tobe bound by any particular theory, the antibodies of the invention mayexert their beneficial effect by reducing pathological tau aggregationand hence the amount of PHF-tau in the brain. The antibodies of theinvention may be used to treat an animal patient belonging to anyclassification. Examples of such animals include mammals such as humans,rodents, dogs, cats and farm animals. For example, the antibodies of theinvention are useful in the preparation of a medicament for treatment ofAD wherein the medicament is prepared for administration in dosagesdefined herein.

Another embodiment of the invention is a method of reducing aggregationof tau in patients in need thereof comprising administering to thepatient a therapeutically effective amount of the isolated antibody ofthe invention for a time sufficient to reduce the aggregation of tau.

Another embodiment of the invention is a method of treating or reducingsymptoms of a neurodegenerative disease that involves aggregation of tauin a patient comprising administering to the patient a therapeuticallyeffective amount of the isolated antibody of the invention for a timesufficient to treat or reduce symptoms of the neurodegenerative disease.

In any of the embodiments above, the neurodegenerative disease thatinvolves aggregation of tau is a tauopathy.

In any of the embodiments above, the isolated antibody comprises anantibody that binds PHF-tau comprising an antigen-binding site of a VHof SEQ ID NO:35 and an antigen-binding site of a VL of SEQ ID NO:36.

In any of the embodiments above, the isolated antibody comprises anantibody that binds PHF-tau comprising an antigen-binding site of a VHof SEQ ID NO:37 and an antigen-binding site of a VL of SEQ ID NO: 38.

As used herein a “tauopathy” encompasses any neurodegenerative diseasethat involves the pathological aggregation of tau within the brain. Inaddition to familial and sporadic AD, other exemplary tauopathies arefrontotemporal dementia with parkinsonism linked to chromosome 17(FTDP-17), progressive supranuclear palsy, corticobasal degeneration,Pick's disease, progressive subcortical gliosis, tangle only dementia,diffuse neurofibrillary tangles with calcification, argyrophilic graindementia, amyotrophic lateral sclerosis parkinsonism-dementia complex,Down syndrome, Gerstmann-Sträussler-Scheinker disease,Hallervorden-Spatz disease, inclusion body myositis, Creutzfeld-Jakobdisease, multiple system atropy, Niemann-Pick disease type C, prionprotein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, postencephalitic parkinsonism, and chronictraumatic encephalopathy, such as dementia pugulistica (boxing disease).(Morris, et al. Neuron 70:410-26, 2011).

A tauopathy-related behavioral phenotype includes cognitive impairments,early personality change and disinhibition, apathy, abulia, mutism,apraxia, perseveration, stereotyped movements/behaviors, hyperorality,disorganization, inability to plan or organize sequential tasks,selfishness/callousness, antisocial traits, a lack of empathy, halting,agrammatic speech with frequent paraphasic errors but relativelypreserved comprehension, impaired comprehension and word-findingdeficits, slowly progressive gait instability, retropulsions, freezing,frequent falls, non-levodopa responsive axial rigidity, supranucleargaze palsy, square wave jerks, slow vertical saccades, pseudobulbarpalsy, limb apraxia, dystonia, cortical sensory loss, and tremor.

Patients amenable to treatment include asymptomatic individuals at riskof AD or other tauopathy, as well as patients presently showingsymptoms. Patients amenable to treatment include individuals who have aknown genetic risk of AD, such as a family history of AD or presence ofgenetic risk factors in the genome. Exemplary risk factors are mutationsin the amyloid precursor protein (APP), especially at position 717 andpositions 670 and 671 (Hardy and Swedish mutations, respectively). Otherrisk factors are mutations in the presenilin genes, PS1 and PS2, andApoE4, family history of hypercholesterolemia or atherosclerosis.Individuals presently suffering from AD can be recognized fromcharacteristic dementia by the presence of risk factors described above.In addition, a number of diagnostic tests are available to identifyindividuals who have AD. These include measurement of cerebrospinalfluid tau and Aβ42 levels. Elevated tau and decreased Aβ42 levelssignify the presence of AD. Individuals suffering from AD can also bediagnosed by AD and Related Disorders Association criteria.

Administration/Pharmaceutical Compositions

Anti-PHF-tau antibodies of the invention are suitable both astherapeutic and prophylactic agents for treating or preventingneurodegenerative diseases that involves pathological aggregation oftau, such as AD or other tauopathies. In asymptomatic patients,treatment can begin at any age (e.g., at about 10, 15, 20, 25, 30years). Usually, however, it is not necessary to begin treatment until apatient reaches about 40, 50, 60, or 70 years. Treatment typicallyentails multiple dosages over a period of time. Treatment can bemonitored by assaying antibody, or activated T-cell or B-cell responsesto the therapeutic agent over time. If the response falls, a boosterdosage is indicated.

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,AD in an amount sufficient to eliminate or reduce the risk, lessen theseverity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presented during development ofthe disease. In therapeutic applications, compositions or medicamentsare administered to a patient suspected of, or already suffering from,such a disease in an amount sufficient to reduce, arrest, or delay anyof the symptoms of the disease (biochemical, histologic and/orbehavioral). Administration of a therapeutic may reduce or eliminatemild cognitive impairment in patients that have not yet developedcharacteristic Alzheimer's pathology. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic and therapeuticregimes, compositions or medicaments are usually administered in severaldosages until a sufficient immune response has been achieved.

Anti-PHF-tau antibodies or fragments thereof of the invention may beadministered in combination with other agents that are effective fortreatment of related neurodegenerative diseases. In the case of AD,antibodies of the invention may be administered in combination withagents that reduce or prevent the deposition of amyloid-beta (Aβ). It ispossible that PHF-tau and Aβ pathologies are synergistic. Therefore,combination therapy targeting the clearance of both PHF-tau and Aβ andAβ-related pathologies at the same time may be more effective thantargeting each individually.

In the case of Parkinson's Disease and related neurodegenerativediseases, immune modulation to clear aggregated forms of the α-synucleinprotein is also an emerging therapy. A combination therapy which targetsthe clearance of both tau and α-synuclein proteins simultaneously may bemore effective than targeting either protein individually.

In the methods of the invention, the “therapeutically effective amount”of the antibody in the treatment or ameliorating symptoms of a tauopathycan be determined by standard research techniques. For example, thedosage of the antibody can be determined by administering the agent torelevant animal models well known in the art.

In addition, in vitro assays can optionally be employed to help identifyoptimal dosage ranges. Selection of a particular effective dose can bedetermined (e.g., via clinical trials) by those skilled in the art basedupon the consideration of several factors. Such factors include thedisease to be treated or prevented, the symptoms involved, the patient'sbody mass, the patient's immune status and other factors known by theskilled artisan. The precise dose to be employed in the formulation willalso depend on the route of administration, and the severity of disease,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The mode of administration for therapeutic use of the antibodies of theinvention may be any suitable route that delivers the agent to the host.Pharmaceutical compositions of these antibodies are useful forparenteral administration, e.g., intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal or intracranialor they can be administered into the cerebrospinal fluid of the brain orspine.

The antibodies of the invention may be prepared as pharmaceuticalcompositions containing an effective amount of the antibody as an activeingredient in a pharmaceutically acceptable carrier. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which theantibody is administered. Such pharmaceutical vehicles can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. For example, 0.4% saline and 0.3% glycine canbe used. These solutions are sterile and generally free of particulatematter. They may be sterilized by conventional, well-known sterilizationtechniques (e.g., filtration). The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the antibodies of the invention in suchpharmaceutical formulation can vary widely, i.e., from less than about0.5%, usually at or at least about 1% to as much as 15 or 20% by weightand will be selected primarily based on required dose, fluid volumes,viscosities, etc., according to the particular mode of administrationselected.

The treatment may be given in a single dose schedule, or as a multipledose schedule in which a primary course of treatment may be with 1-10separate doses, followed by other doses given at subsequent timeintervals required to maintain and or reinforce the response, forexample, at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. Examples of suitable treatment schedulesinclude: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii)0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient toelicit the desired responses expected to reduce disease symptoms, orreduce severity of disease.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg, about 50 ng to about 30 mg or about5 mg to about 25 mg of an antibody of the invention. Similarly, apharmaceutical composition of the invention for intravenous infusioncould be made up to contain about 250 ml of sterile Ringer's solution,and about 1 mg to about 30 mg or about 5 mg to about 25 mg of anantibody of the invention. Actual methods for preparing parenterallyadministrable compositions are well known and are described in moredetail in, for example, “Remington's Pharmaceutical Science”, 15th ed.,Mack Publishing Company, Easton, Pa.

The antibodies of the invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with antibody and other protein preparationsand art-known lyophilization and reconstitution techniques can beemployed.

Diagnostic Methods and Kits

Antibodies of the invention may be used in methods of diagnosing AD orother tauopathy in a subject. This method involves detecting, in thesubject, the presence of PHF-tau using a diagnostic reagent such as anantibody or a fragment thereof of the present invention.

PHF-tau may be detected in a biological sample from a subject (e.g.,blood, urine, cerebral spinal fluid) by contacting the biological samplewith the diagnostic antibody reagent, and detecting binding of thediagnostic antibody reagent to PHF-tau in the sample from the subject.Assays for carrying out the detection include well known methods such asELISA, immunohistochemistry, western blot, or in vivo imaging. Exemplarydiagnostic antibodies are antibodies PT1 and PT3 of the invention, andare of IgG1, κ type.

Diagnostic antibodies or similar reagents can be administered byintravenous injection into the body of the patient, or directly into thebrain by any suitable route that delivers the agent to the host asexemplified above. The dosage of antibody should be within the sameranges as for treatment methods. Typically, the antibody is labeled,although in some methods, the primary antibody with affinity for PHF-tauis unlabelled and a secondary labeling agent is used to bind to theprimary antibody. The choice of label depends on the means of detection.For example, a fluorescent label is suitable for optical detection. Useof paramagnetic labels is suitable for tomographic detection withoutsurgical intervention. Radioactive labels can also be detected using PETor SPECT.

Diagnosis is performed by comparing the number, size, and/or intensityof labeled PHF-tau, tau aggregates, and/or neurofibrillary tangles in asample from the subject or in the subject, to corresponding baselinevalues. The baseline values can represent the mean levels in apopulation of undiseased individuals. Baseline values can also representprevious levels determined in the same subject.

The diagnostic methods described above can also be used to monitor asubject's response to therapy by detecting the presence of PHF-tau in asubject before, during or after the treatment. A decrease in valuesrelative to baseline signals a positive response to treatment. Valuescan also increase temporarily in biological fluids as pathological tauis being cleared from the brain.

The present invention is further directed to a kit for performing theabove described diagnostic and monitoring methods. Typically, such kitscontain a diagnostic reagent such as the antibodies of the invention,and optionally a detectable label. The diagnostic antibody itself maycontain the detectable label (e.g., fluorescent molecule, biotin, etc.)which is directly detectable or detectable via a secondary reaction(e.g., reaction with streptavidin). Alternatively, a second reagentcontaining the detectable label may be utilized, where the secondreagent has binding specificity for the primary antibody. In adiagnostic kit suitable for measuring PHF-tau in a biological sample,the antibodies of the kit may be supplied prebound to a solid phase,such as to the wells of a microtiter dish.

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Example 1 Purification of Paired Helical Filament-Tau (PHF-Tau)

PHF-tau was partially purified by a modified method of Greenberg andDavies (Greenberg and Davies Proc Natl Acad Sci USA 87:5827-31, 1990).Briefly, postmortem tissue from the cortex obtained from ahistologically confirmed Alzheimer patient was partially purified.Typically, 5 mg of frontal cortex was homogenized in 10 vol of coldbuffer Buffer H (10 mM Tris, 800 mM NaCl, 1 mM EGTA and 10% sucrose/pH7.4) using a glass/Teflon Potter tissue homogenizer (IKA Works, Inc;Staufen, Germany) at 1000 rpm. The homogenized material was centrifugedat 27000 g for 20 min in a Sorvall rotor SS34. The pellet was discardedand the supernatant was adjusted to a final concentration of 1% (w/v)N-lauroylsarcosine and 1% (v/v) 2-mercaptoethanol and incubated for 2 hat 37° C. Subsequently the supernatant was centrifuged at 108000 g for35 min at 20° C. in a Beckman 60Ti rotor. The pellet was carefullywashed in PBS and suspended in PBS. The supernatant was centrifuged asecond time as described and the final pellet was dissolved, aliquotedand frozen at −80° C. The quality of the PHF-tau preparations wereevaluated on a 12% SDS-PAGE and western blot with anti-tau antibodiesAT8 and HT7 (ThermoScientific, Rockford, Ill.). AT8 detects PHF-tauphosphorylated at 5202/T205, but does not bind to non-phosphorylatedPHF-tau nor to the wild type tau. HT7 binds to a non-phosphorylatedepitope at Tau amino acids 159-163 (of SEQ ID NO: 6), and recognizesboth tau and PHF-tau. A good quality PHF-tau preparation is composed of4 bands having molecular weights of about 60, 64, 66 and 72 kDa on aWestern blot detected with an antibody reactive with hyperphosphorylatedPHF-tau such as AT8. Two separate PHF-tau preparations with comparablequality and purity were made from the same brain sample. Preparation 1was used for immunization.

Example 2 Generation of Monoclonal Antibodies Against PHF-Tau

Anti-PHF-tau antibodies were generated using standard hybridomatechnology in normal Balb/c mice (Kohler and Milstein Nature 256:495-7,1975). Obtained hybridomas were seeded in 96-well plates and screenedafter 10 days in a direct ELISA on 25 ng/well coated PHF-tau asdescribed below. Positive cells were tested for cross-reactivity on 10ng/well coated with control tau (SEQ ID NO:6) expressed in E. Coli BL21cells and purified by heat treatment and ammonium sulphateprecipitation, and were immediately subcloned and positive clones werefrozen in liquid nitrogen. All hybridomas were grown in Dulbecco'smodified Eagle's medium supplemented with 10% foetal calf serum(Hyclone, Europe), Hybridoma Fusion Cloning Supplement (2%) (Roche,Brussels, Belgium) 2% HT (Sigma, USA), 1 mM sodium pyruvate, 2 mML-glutamine and penicillin (100 U/ml) and Streptomycin (50 mg/ml).

Antibody variable regions were cloned from select hybridoma cells ontomouse IgG1/IgG2/κ background and expressed and purified using routinemethods.

Direct ELISA for Antibody Selection

25 ng/well PHF-tau was coated overnight at 4° C. in NUNC Maxisorp (LifeTechnologies) flat-bottom high-binding 96-well micro titer plates in 50μl/well coating buffer (10 mM Tris, 10 mM NaCl, and 10 mM NaN3, pH 8.5).The next day, the plates were blocked with 75 μl/well of 0.1% casein inPBS for 60 min at room temperature. Next, 50 μl hybridoma supernatantwas added and incubated for 1 h at 37° C. After washing, the boundmonoclonal antibodies were detected with 50 μl/well of Sheep-anti-mouseIgG conjugated with horseradish peroxidase for 1 hr at 37° C.(Amersham-Pharmacia Biotech). Both reagents were diluted in 0.1%Casein/PBS. The plates were washed and 50 μl of a solution of 0.42 mM3,5,3′,5′-tetramethyl-benzidine, 0.003% (v/v) H₂O₂ in 100 mM citric acidand 100 mM disodium hydrogen phosphate (pH 4.3) was added as thesubstrate. The reaction was allowed to proceed for maximum 15 min on aplate shaker at room temperature, after which the color development wasstopped with 2 N H₂SO₄, 50 μl/well and the plates, were read on a microtiter plate reader at 450 nm (Thermomax, Molecular Devices).

Specificity of the Monoclonal Antibodies

Select antibodies obtained from the hybridoma screen were tested fortheir cross-reactivity with recombinantly expressed control tau (SEQ IDNO:6). 500 ng of PHF-tau and 200 ng of control tau were loaded on aNuPAGE® Novex® Bis-Tris 4-12% gel and blotted on a nitrocellulosemembrane by use of an iBlot system (Invitrogen), according to themanufacturer's instructions. Membranes were blocked for 1 hour withTris-Buffered Saline Tween-20 (TBS-T; 1M Tris, 150 mM NaCl and 0.05%Tween-20, pH 8.5) containing non fat dry milk (NFDM) (5% w/v; Biorad)and washed three times in TBS-T. Incubation with the primary controlantibodies (1 μg/ml) HT7, AT8, AT100 (ThermoScientific, Rockford, Ill.),and BT2 diluted in TBS-T containing NFDM (5% w/v) was overnight at 4° C.PT1, PT2, PT3, PT4 and PT5 monoclonal antibodies selected from thehybridoma screen were added in culture supernatant containing 10% FCS.Primary antibodies were detected using Sheep-anti-mouse Ig conjugatedwith HRPO (1:20000 in TBS-T, Amersham Biosciences) via West Dura®enhanced chemiluminescence (Pierce, Thermoscientific). Signals werecaptured by the Lumi-imaging system (Roche Diagnostic). PT1 and PT3reacted with PHF-tau and did not react with control tau in westernblots. PT2 was reactive with both proteins. Binding profiles of HT7 andAT8 are described above. AT100 binds to phosphorylated Ser212/Thr214 andbinds to PHF-tau but not wild type tau. BT2 recognizes anon-phosphorylated epitope comprising S199/S202, and thus recognizeswild type tau but not PHF-tau.

Competitive Epitope Binding

Monoclonal antibodies PT1, PT2, PT3, PT4, PT5, AT8 (ThermoScientific,Rockford, Ill.), AT100 (ThermoScientific, Rockford, Ill.) and HT7(MN1000) (Thermo Scientific, Rockford, Ill.) were evaluated forcompetitive binding to PHF-tau or phosphorylated tau peptides.Antibodies were labeled using MSD® SULFO-TAG NHS Ester (Meso ScaleDiscovery) according to the manufacturer's instruction

For competition with labeled PT1, PT3, AT100 and HT7, 5 μL (50μg/mL)/well of enriched PHF-Tau proteins (purified as described above)was coated on MSD HighBind plate (Meso Scale Discovery, Gaithersburg,Md.) for 2 hr at room temperature (RT). For competition with AT8, 25 μLof 0.1 mg/well synthetic biotinylated and pegylated peptideRSGYSSPG(pS)PG(pT)PGSRSR-OH (New England Peptide, LLC., Gardner, Mass.)(SEQ ID NO:39) corresponding to residues 194-211 of control tau (SEQ IDNO:6) phosphorylated at residues corresponding to 5202/T205 in controltau was coated on Streptavidin-charged plates (Meso Scale Discovery,Gaithersburg, Md.). After coating, wells were blocked with 150 μL of 5%MSD Blocker A buffer at RT for 2 hr, and washed three times with 0.1 MHEPES buffer, pH 7.4. 25 μL of a mixture of labeled individual anti-tauantibody (10 nM or 50 nM) and serial dilutions of various unlabeledcompetitor antibodies (1 nM to 2 μM) was added on each well. The plateswere incubated for 2 hours at RT with gentle shaking and washed 3 timesas above. 150 μL/well of diluted MSD Read Buffer T was added and theplates were read in a SECTOR Imager 6000 (Meso Scale Discovery,Gaithersburg, Md.).

Non-overlapping epitopes of anti-Tau mAbs HT7, AT100 and AT8 aredescribed above. Based on the published data, these antibodies were notexpected to compete with each other in binding to Tau proteins orpeptides.

Based on competition assays performed, none of the antibodies competedwith each other for binding, indicating that they all bind to differentepitopes. In each experiment, only self-inhibition was observed. FIGS.1-5 show results of competition assays with labeled AT8, PT1, PT3,AT100, and HT7, respectively.

Example 3 Anti-PHF Tau Antibodies Reduce PHF-Tau Accumulation In Vivo

5-month old female P301L mice (Taconic, cat#002508) were treated onceweekly with mouse IgG1, saline, PT3 (500 μg/mouse) or AT8 (expressedfrom hybridoma ECACC, deposit number 9110086) for 5 months. Mice wereanesthetized, perfused with cold PBS and the brains dissected on ice.For each mouse, one brain hemisphere was homogenized in 10 volume of H—buffer, followed by centrifugation at 21,000 g for 20 min at 4° C. Theresulting supernatant was further centrifuged at 100,000 g for 60 minAfter centrifugation, the pellet (fraction P1) was recovered andre-suspended with lysis buffer for Western and ELISA analyses, asdescribed by Chai et al. J Biol Chem 286:34457-67, 2011. For cortexsamples, the fraction P1 was further treated with 1% (w/v)N-lauroylsarcosine and ultracentrifuged to further enrich PHF-tau in thepellet. Male P301L mice were found to have low transgene expression andwere not included in the analyses.

Phosphorylated tau was measured in brainstem homogenates (fraction P1)with sandwich ELISA using antibodies AT8 and AT100 as capture antibodiesfollowed by detection by biotinylated HT7 and avidin-HRP (FIGS. 6A and6B), and with AT100 in a western blot (FIG. 6C) essentially as describedin Chai et al. J Biol Chem 286:34457-67, 2011. Briefly, the P1 pelletwas resuspended with lysis buffer (Cell Signaling). P1 pellet sampleswere incubated in AT8 or AT100 (Thermo Scientific) pre-coated wells andthe biotin labeled HT-7 antibody. Samples were then washed 5 times withbuffer, followed by incubation with avidin-HRP for one hour. Followingthis, samples were incubated with one step TMB substrate (ThermoScientific) for 30 min, followed by 2N H₂SO₄. Finally, the reaction wasread at 450 nm and the quantity of AT8- or AT100-reactive tau in brainwas determined using a standard curve derived from human AD brainhomogenates, and plotted as a relative amount of AD brain homogenate(ng/ml) providing the same ELISA signal as an average samples from anon-transgenic animal (B6).

A statistically significant decrease or trend towards significance inphosphorylated tau was seen in P301L transgenic animals treated with PT3when compared to the isotype control administered animals in ELISAassays using either AT100 (p=0.057) or AT8 (p=0.0475) to detectedphosphorylation (ELISA signal: Saline group (1135±228.8); IgG1 group(1344±245.6); PT3 group (660.5±134.5); AT8 group (1271±274)).

To confirm the data obtained with ELISA, brainstem homogenates (fractionP1) from IgG1- or PT3-treated animals were analyzed on western blot bydetecting PHF-tau using AT100 antibody. The filters were blotted usinganti-actin antibody as a loading control (FIG. 6B). Western blots showedattenuated PHF-tau amount detected with AT100 antibody when compared tothe animals treated with IgG1.

For cortex analysis, both sarcosyl soluble (representing soluble tau)and insoluble (representing PHF-tau) cortex fractions were analyzed bysandwich ELISA using a pan-tau antibody (PT4) or phospho-tau antibody(AT8) for capturing followed by a biotin-pan-tau antibody (hTau10)followed by HRP-avidin. Animals treated with PT3 had similar levels oftotal tau in comparison to animals treated with the isotype controlIgG1. A trend towards lower PHF-tau levels were evident in animalstreated with PT3 when compared to the isotype control in theN-lauroylsarcosine insoluble fractions (ELISA capture with PT4: IgGgroup: 851026±261198 and PT3 group: 585639±120498; ELISA, capture withAT8: IgG group: 1125886±286240 (N=10) and pT3 group: 746582±124970(N=7)).

Example 4 Characterization of Anti-PHF-Tau Antibodies AffinityDetermination

The interactions of monoclonal antibodies PT1 and PT3 with recombinanthuman soluble tau or PHF-tau were studied by ProteOn. All interactionswere studied at 25° C. using PBS pH 7.4, supplemented with 3 mM EDTA,and 0.005% Tween-20 as running or system buffer. Two differentexperiment formats were used, one for the interaction with recombinantlyexpressed control tau and another for the interaction with PHF-tau. Inthese experiments HT7 (Pierce, cat #MN1000), a mouse anti-tau antibodywas used as a positive control.

To study the interaction with recombinantly expressed control tau abiosensor surface was prepared by coupling an anti-human or anti-mouseIgG Fcγ fragment specific antibody (Ab) to the surface of a GLC(ProteOn) sensor chip using each manufacturer's instructions foramine-coupling chemistry (5000 response units (RU)). The coupling bufferwas 10 mM sodium acetate, pH 4.5. The anti-PHF-tau antibodies werediluted in the running buffer and injected to obtain a capture of 60-130RUs. Capture of anti-PFH-Tau mAbs was followed by injection ofrecombinantly expressed control tau (Tau-441, Sigma catalog# T0576-50ug) in solution (0.1 to 75 nM in 5-fold dilutions). The association wasmonitored for 2 minutes (80 μL injected at 40 μL/min) The dissociationwas monitored for 10 minutes. Regeneration of the sensor surface wasobtained with 0.85% H₃PO₄, or 0.85% H₃PO₄ followed by 50 mM NaOH. Thedata were fit using a 1:1 binding model. The data were fit using a 1:1binding model.

TABLE 2 mAb Antigen k_(on) (M⁻¹ s⁻¹) k_(off) (s⁻¹) *K_(D) (pM) PT1Control ** Tau PT1 PHF-Tau 2.01E+05 6.47E−05 322 PT3 Control *** Tau PT3PHF-Tau (1.23 ± 0.06)E+06 (2.18 ± 0.28)E−05 18 ± 2 *For PHF-tau this isthe apparent intrinsic affinity where KD is obtained as the ratio ofkffl/kon-1 derived from a fit performed using a bivalent binding model.** No significant binding *** No binding in 4 of 5 experiments

To study the interaction with PHF-tau a biosensor surface was preparedby capture-coupling PHF-tau using HT7 as the capture reagent. Additionalpreparation of the PHF-tau as described earlier was required for ProteOnto limit the amount of insoluble material entering the fluidics. PHF-tauas described above was additionally prepared by 2-times centrifugationat 5000 g, 5° C., 10 min where the supernatant from the secondcentrifugation was then diluted 1/20 or 1/40 in running buffer. Toprepare the chip, HT7 was covalently immobilized to the surface of a GLC(ProteOn) sensor chip using each manufacturer's instructions foramine-coupling chemistry (˜3000 response units (RU). The coupling bufferwas 10 mM sodium acetate, pH 4.5. After HT7 immobilization PHF-tau wasinjected and captured (˜300 RU) by HT7. After capture, PHF-tau wascovalently immobilized to the sensor chip by activation of the chipusing each manufacturer's instructions for amine-coupling chemistry.Remaining reactive sites were finally blocked by injection ofethanolamine. After preparation and stabilization of thePHF-tau-modified surface and reference surface (containing no antigen),the anti-PHF-tau antibodies were diluted in the running buffer andinjected in solution (0.1-75 nM in 5-fold dilutions). The associationwas monitored for 3 minutes (120 μL, injected at 40 μL/min) Thedissociation was monitored for 10 or 15 minutes. Regeneration of thesensor surface was obtained with 10 mM Gly pH 2. The data were fit usinga bivalent binding model where the apparent intrinsic affinity wasreported as the ratio of koff-1/kon-1.

Based on the ProteOn experiments PT1 bound PHF-tau with 322 pM affinityand showed no binding to control tau under the conditions tested (Table2). PT3 bound PHF-tau with 18±2 pM affinity and showed no binding tocontrol tau in 4 out of 5 measurements under the conditions tested. Oneout of the 5 ProteOn measurements showed weak binding which could beused to estimate affinity >75 nM on the basis of the highestconcentration of control tau used.

The present invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

We claim:
 1. An isolated antibody that binds PHF-tau comprising anantigen-binding site of a heavy chain variable region (VH) of SEQ IDNO:35 or 37, and an antigen-binding site of a light chain variableregion (VL) of SEQ ID NO:36 or
 38. 2. The isolated antibody of claim 1,wherein the antigen-binding site of the VH comprises heavy chaincomplementarity determining regions (CDRs) 1 (HCDR1), 2 (HCDR2) and 3(HCDR3) of SEQ ID NOs:7, 8 and 9 or SEQ ID NOs:13, 14 and 15,respectively, and the antigen-binding site of the VL comprises lightchain CDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs:10, 11 and12 or SEQ ID NOs: 16, 17 and 18, respectively.
 3. The isolated antibodyof any one of claim 1 or 2 that binds PHF-tau comprising anantigen-binding site of a VH of SEQ ID NO:35 and an antigen-binding siteof a VL of SEQ ID NO:36.
 4. The isolated antibody of claim 3, whereinthe antigen-binding site of the VH comprises HCDR1, HCDR2 and HCDR3 ofSEQ ID NOs:7, 8 and 9, respectively, and the antigen-binding site of theVL comprises LCDR1, LCDR2 and LCDR3 of SEQ ID NOs:10, 11 and 12,respectively.
 5. The isolated antibody of any one of claim 1 or 2 thatbinds PHF-tau comprising an antigen-binding site of a VH of SEQ ID NO:37and an antigen-binding site of a VL of SEQ ID NO:
 38. 6. The isolatedantibody of claim 5, wherein the antigen-binding site of the VHcomprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs:13, 14 and 15,respectively, and the antigen-binding site of the VL comprises LCDR1,LCDR2 and LCDR3 of SEQ ID NOs:16, 17 and 18, respectively.
 7. Theisolated antibody of any one of claims 1-6, wherein the antibody ishumanized.
 8. An isolated antibody or fragment that competes for PHF-taubinding with a monoclonal antibody comprising an antigen-binding site ofa VH of SEQ ID NO:35 and an antigen-binding site of a VL of SEQ IDNO:36, or an antigen-binding site of a VH of SEQ ID NO:37 and anantigen-binding site of a VL of SEQ ID NO:38.
 9. An isolatedpolynucleotide encoding an antibody VH comprising HCDR1, HCDR2 and HCDR3of SEQ ID NOs:7, 8 and 9, respectively, or SEQ ID NOs:13, 14 and 15,respectively.
 10. An isolated polynucleotide encoding an antibody VLcomprising LCDR1, LCDR2 and LCDR3 of SEQ ID NOs:10, 11 and 12,respectively, or SEQ ID NOs:16, 17 and 18, respectively.
 11. A vectorcomprising a polynucleotide of claim 9 or
 10. 12. A host cell comprisingthe vector of claim
 11. 13. A method of making an antibody that bindsPHF-tau comprising culturing the host cell of claim 12 and recoveringthe antibody produced by the host cell.